Animals
Twenty-four male C57/BL6 mice (aged 6 weeks), which were housed in a specific-pathogen-free (SPF) facility, were obtained from experimental animal center of Shanxi Medical University (license number: SCXK (Jin) 2015-0001, Taiyuan, China). All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Shanxi Medical University in China.
Animal experiment
All mice were randomly divided into three groups after 1 week of adaptive feeding as follows: NS (normal saline) group (n=6), AIH+NS group (n=6) and AIH+RAP group (n=6). The AIH mouse model was established as previously reported [1-3] by intraperitoneal administration of 0.5 ml fresh S-100 antigen emulsified in an equal volume of complete Freund’s adjuvant (CFA, Sigma, Cat No.:F5881, USA) (the concentration of S-100 antigen is 1 mg/mL) on 1st and 7th day. On 4th day, the AIH+NS group and AIH+RAP group were given 0.2mL intraperitoneal injection with normal saline (NS) and rapamycin (Sangon, Cat No.:A606203, China, 1 mg/kg), respectively, once a day for 20 days. All experimental animals were anesthetized by intraperitoneal injection with sodium pentobarbital (60 mg/kg). After deep anesthesia, cervical dislocation was performed and euthanasia was performed. Animal studies were approved by the Animal Use and Care Committee of Shanxi medical university, and were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals.
Biochemical indexes
On 24th day, blood was drawn from retro-orbital plexus and sera were collected by centrifugation (4 °C, 3000rpm, 15 min) and stored at −80 °C. The serum samples were analyzed for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by automatic biochemical analyzer.
Histopathology, TUNEL and Immunohistochemistry assay
Liver tissues were fixed with 10% neutral formalin and embedded in paraffin wax and sliced. The TUNEL Apoptosis Assay Kit (Beyotime, Cat No.:C1086, China) was used to detect the apoptosis of liver, according to the manufacturer’s instruction. The slides were stained with hematoxylin-eosin (HE). The degree of inflammation of the liver was graded as follows: grade 0 = normal liver tissue; grade 1 = mild infiltration of inflammatory cells with rare hepatic cells necrosis; grade 2 = medium damage of hepatic cells accompanied by infiltration of inflammatory cells and regional hepatic cell necrosis; grade 3 = wide range inflammatory cells infiltration in portal area and hepatic lobules accompanied with wide range hepatic cells necrosis. Three randomly selected fields were scored by three individuals in a blinded manner. Liver Sections deparaffinization, rehydration, blockade of endogenous peroxidase, and antigen retrieval were sequentially performed, and then incubated with primary antibody anti-IFN-γ(1:100; Bioss, Cat No.:bs-0481R, China), or anti-IL-17(1:100; Bioss, Cat No.:bs-1183R, China) at 4°C overnight. After being incubated in secondary antibody (1:500, Boster, Cat No.:BA1041, China), the sections were then colorized by diaminobenzidine reaction.
Liver tissue lymphocytes isolation
Liver tissues were dissected into small pieces, ground and filtered through stainless steel meshes. Liver lymphocytes were isolated from the cell suspensions by gradient centrifugation at 1,400×g for 25 min with Percoll (Solarbio, Cat No.:P8370, China) and lymphocytes were suspended in RPMI 1640 media. The proportion of living cells is verified by trypan blue dye, which is greater than 95%.
Intracellular cytokine staining and Flow cytometry
For measuring intracellular amount of IFN-γ and IL-17 positive cells, liver lymphocytes (5×106 cells) were added to 6-well plate. Firstly, cells were incubated and stimulated with phorbolmyristate acetate (PMA, Sigma, Cat No.:P1585, USA, 50 ng/ml), ionomycin (Beyotime, Cat No.:S1672, China, 5 μg/ml) and brefeldin A (BD, Cat No.:555029, USA, 20 μg/ml) at 37 °C for 6 h. Following fixation and permeabilization, the cultured cells were stained with anti-CD4 antibody (1:5000, Cat BD, No.:553654, USA) anti-IFN-γ (1:5000, BD, Cat No.:560660, USA), and anti-IL-17 (1:5000, BD, Cat No.:560821, USA) for 30 min. Flow cytometry was used to detect the frequency of Th1 and Th17 cells.
Western blotting
Briefly, liver tissues were homogenized in RIPA lysis buffer (Boster, Cat No.:AR0102, China) containing a protease inhibitor cocktail (Boster Cat No.:AR1183, China). Then, the protein concentrations were detected by BCA protein assay kit (Boster Cat No.:AR1189, China) according to the manufacturer's instruction. Protein extracts were separated on SDS-PAGE gels and further electro-transferred to NC membranes (Boster Cat No.:AR0135-04, China). After being blocked with 5% nonfat skim milk, the membranes were incubated overnight at 4 °C with appropriate primary antibodies against p70s6k (1:1000, CST, Cat No.:2708, USA), phospho-p70s6k (Thr421/Ser424, 1:1000, CST, Cat No.:9204, USA), STAT3 (1:2000; CST, Cat No.:12640, USA), phospho-STAT3 (Tyr705, 1:2000, CST, Cat No.:9145, USA), respectively. Membranes were then washed with TBST and incubated with peroxidase-conjugated secondary antibody (1:5000, Beyotime, Cat No.:A0208, China) for 1 h at room temperature. Finally, the bands on the membranes were visualized and analyzed by electrochemiluminescence detection. The β-actin staining (1:2000, Bioworld, Cat No.:AP0714, China) was used as internal control.
Statistical analysis
All data were analyzed using SPSS17.0 software (SPAA Inc., USA). Results were expressed as mean ±standard deviation and one-way analysis of variance was used. Comparison among groups used LSD analysis (homogeneity of variance) or Dunnett’s T3 (heterogeneous variance). P <0.05 was statistically significant.